Novel low molecular weight cationic lipids for oligonucleotide delivery

ABSTRACT

The instant invention provides for novel cationic lipids that can be used in combination with other lipid components such as cholesterol and PEG-lipids to form lipid nanoparticles with oligonucleotides. It is an object of the instant invention to provide a cationic lipid scaffold that demonstrates enhanced efficacy along with lower liver toxicity as a result of lower lipid levels in the liver. The present invention employs low molecular weight cationic lipids with one short lipid chain to enhance the efficiency and tolerability of in vivo delivery of siRNA.

BACKGROUND OF THE INVENTION

The present invention relates to novel cationic lipids that can be usedin combination with other lipid components such as cholesterol andPEG-lipids to form lipid nanoparticles with oligonucleotides, tofacilitate the cellular uptake and endosomal escape, and to knockdowntarget mRNA both in vitro and in vivo.

Cationic lipids and the use of cationic lipids in lipid nanoparticlesfor the delivery of oligonucleotides, in particular siRNA and miRNA,have been previously disclosed. Lipid nanoparticles and use of lipidnanoparticles for the delivery of oligonucleotides, in particular siRNAand miRNA, has been previously disclosed. Oligonucleotides (includingsiRNA and miRNA) and the synthesis of oligonucleotides has beenpreviously disclosed. (See US patent applications: US 2006/0083780, US2006/0240554, US 2008/0020058, US 2009/0263407 and US 2009/0285881 andPCT patent applications: WO 2009/086558, WO2009/127060, WO2009/132131,WO2010/042877, WO2010/054384, WO2010/054401, WO2010/054405 andWO2010/054406). See also Semple S. C. et al., Rational design ofcationic lipids for siRNA delivery, Nature Biotechnology, publishedonline 17 Jan. 2010; doi:10.1038/nbt.1602.

Other cationic lipids are disclosed in US patent applications: US2009/0263407, US 2009/0285881, US 2010/0055168, US 2010/0055169, US2010/0063135, US 2010/0076055, US 2010/0099738 and US 2010/0104629.

Traditional cationic lipids such as CLinDMA and DLinDMA have beenemployed for siRNA delivery to liver but suffer from non-optimaldelivery efficiency along with liver toxicity at higher doses. It is anobject of the instant invention to provide a cationic lipid scaffoldthat demonstrates enhanced efficacy along with lower liver toxicity as aresult of lower lipid levels in the liver. The present invention employslow molecular weight cationic lipids with one short lipid chain toenhance the efficiency and tolerability of in vivo delivery of siRNA.

SUMMARY OF THE INVENTION

The instant invention provides for novel cationic lipids that can beused in combination with other lipid components such as cholesterol andPEG-lipids to form lipid nanoparticles with oligonucleotides. It is anobject of the instant invention to provide a cationic lipid scaffoldthat demonstrates enhanced efficacy along with lower liver toxicity as aresult of lower lipid levels in the liver. The present invention employslow molecular weight cationic lipids with one short lipid chain toenhance the efficiency and tolerability of in vivo delivery of siRNA.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: LNP (Compound 1) efficacy in mice.

FIG. 2: LNP (Compound 1) efficacy in rat.

FIG. 3: LNP (Compound 1) LFT elevations in rat.

FIG. 4: Cationic lipid (Compound 1) liver levels in rat.

DETAILED DESCRIPTION OF THE INVENTION

The various aspects and embodiments of the invention are directed to theutility of novel cationic lipids useful in lipid nanoparticles todeliver oligonucleotides, in particular, siRNA and miRNA, to any targetgene. (See US patent applications: US 2006/0083780, US 2006/0240554, US2008/0020058, US 2009/0263407 and US 2009/0285881 and PCT patentapplications: WO 2009/086558, WO2009/127060, WO2009/132131,WO2010/042877, WO2010/054384, WO2010/054401, WO2010/054405 andWO2010/054406). See also Semple S. C. et al., Rational design ofcationic lipids for siRNA delivery, Nature Biotechnology, publishedonline 17 Jan. 2010; doi:10.1038/nbt.1602.

The cationic lipids of the instant invention are useful components in alipid nanoparticle for the delivery of oligonucleotides, specificallysiRNA and miRNA.

In a first embodiment of this invention, the cationic lipids areillustrated by the Formula A:

wherein:

R¹ and R² are independently selected from H, (C₁-C₆)alkyl, heterocyclyl,and polyamine, wherein said alkyl, heterocyclyl and polyamine areoptionally substituted with one to three substituents selected from R′,or R¹ and R² can be taken together with the nitrogen to which they areattached to form a monocyclic heterocycle with 4-7 members optionallycontaining, in addition to the nitrogen, one or two additionalheteroatoms selected from N, O and S, said monocyclic heterocycle isoptionally substituted with one to three substituents selected from R′;

R′ is independently selected from halogen, R″, OR″, SR″, CN, CO₂R″ orCON(R″)₂;

R″ is independently selected from H and (C₁-C₆)alkyl, wherein said alkylis optionally substituted with halogen and OH;

L₁ is selected from C₄-C₂₂ alkyl and C₄-C₂₂ alkenyl, said alkyl andalkenyl are optionally substituted with one or more substituentsselected from R′; and

L₂ is selected from C₃-C₁₃ alkyl and C₃-C₁₃ alkenyl, said alkyl andalkenyl are optionally substituted with one or more substituentsselected from R′;

or any pharmaceutically acceptable salt or stereoisomer thereof.

In a second embodiment, the invention features a compound having FormulaA, wherein:

R¹ and R² are each methyl;

L₁ is selected from C₄-C₂₂ alkyl and C₄-C₂₂ alkenyl; and

L₂ is selected from C₃-C₁₃ alkyl and C₃-C₁₃ alkenyl;

or any pharmaceutically acceptable salt or stereoisomer thereof.

Specific cationic lipids are:

-   R—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine    (Compound 2);-   S—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine    (Compound 1);-   1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine    (Compound 3);-   (2S)—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine    (Compound 4);-   1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine    (Compound 5);-   (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine    (Compound 6);-   (2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine    (Compound 7);-   N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine    (Compound 8);-   N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-amine    (Compound 9);-   (2S)—N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine    (Compound 10);-   (2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine    (Compound 11);-   (2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine    (Compound 12);-   1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine    (Compound 13);-   1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine    (Compound 14);-   (2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine    (Compound 15);-   (2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine    (Compound 16);-   1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine    (Compound 17);-   1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine    (Compound 18);-   (2R)—N,N-dimethyl-1-[(1-methyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine    (Compound 19);-   (2R)-1[(3,7-dimethyloctypoxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine    (Compound 20);-   N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine    (Compound 21); and-   N,N-dimethyl-1-{[8-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine    (Compound 22);    or any pharmaceutically acceptable salt or stereoisomer thereof.

In another embodiment, the cationic lipids disclosed are useful in thepreparation of lipid nanoparticles.

In another embodiment, the cationic lipids disclosed are usefulcomponents in a lipid nanoparticle for the delivery of oligonucleotides.

In another embodiment, the cationic lipids disclosed are usefulcomponents in a lipid nanoparticle for the delivery of siRNA and miRNA.

In another embodiment, the cationic lipids disclosed are usefulcomponents in a lipid nanoparticle for the delivery of siRNA.

The cationic lipids of the present invention may have asymmetriccenters, chiral axes, and chiral planes (as described in: E. L. Elieland S. H. Wiley, Stereochemistry of Carbon Compounds, John Wiley & Sons,New York, 1994, pages 1119-1190), and occur as racemates, racemicmixtures, and as individual diastereomers, with all possible isomers andmixtures thereof, including optical isomers, being included in thepresent invention. In addition, the cationic lipids disclosed herein mayexist as tautomers and both tautomeric forms are intended to beencompassed by the scope of the invention, even though only onetautomeric structure is depicted.

It is understood that substituents and substitution patterns on thecationic lipids of the instant invention can be selected by one ofordinary skill in the art to provide cationic lipids that are chemicallystable and that can be readily synthesized by techniques known in theart, as well as those methods set forth below, from readily availablestarting materials. If a substituent is itself substituted with morethan one group, it is understood that these multiple groups may be onthe same carbon or on different carbons, so long as a stable structureresults.

It is understood that one or more Si atoms can be incorporated into thecationic lipids of the instant invention by one of ordinary skill in theart to provide cationic lipids that are chemically stable and that canbe readily synthesized by techniques known in the art from readilyavailable starting materials.

In the compounds of Formula A, the atoms may exhibit their naturalisotopic abundances, or one or more of the atoms may be artificiallyenriched in a particular isotope having the same atomic number, but anatomic mass or mass number different from the atomic mass or mass numberpredominantly found in nature. The present invention is meant to includeall suitable isotopic variations of the compounds of Formula A. Forexample, different isotopic forms of hydrogen (H) include protium (¹H)and deuterium (²H). Protium is the predominant hydrogen isotope found innature. Enriching for deuterium may afford certain therapeuticadvantages, such as increasing in vivo half-life or reducing dosagerequirements, or may provide a compound useful as a standard forcharacterization of biological samples. Isotopically-enriched compoundswithin Formula A can be prepared without undue experimentation byconventional techniques well known to those skilled in the art or byprocesses analogous to those described in the Scheme and Examples hereinusing appropriate isotopically-enriched reagents and/or intermediates.

As used herein, “alkyl” means a straight chain, cyclic or branchedsaturated aliphatic hydrocarbon having the specified number of carbonatoms.

As used herein, “alkenyl” means a straight chain, cyclic or branchedunsaturated aliphatic hydrocarbon having the specified number of carbonatoms including but not limited to diene, triene and tetraeneunsaturated aliphatic hydrocarbons.

Examples of a cyclic “alkyl” or “alkenyl are:

As used herein, “heterocyclyl” or “heterocycle” means a 4- to10-membered aromatic or nonaromatic heterocycle containing from 1 to 4heteroatoms selected from the group consisting of O, N and S, andincludes bicyclic groups. “Heterocyclyl” therefore includes, thefollowing: benzoimidazolyl, benzofuranyl, benzofurazanyl,benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl,carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl,indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl,isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl,oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl,pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl,pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl,tetrahydropyranyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl,thiazolyl, thienyl, triazolyl, azetidinyl, 1,4-dioxanyl,hexahydroazepinyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl,thiomorpholinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl,dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl,dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl,dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl,dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, andN-oxides thereof all of which are optionally substituted with one tothree substituents selected from R″.

As used herein, “polyamine” means compounds having two or more aminogroups. Examples include putrescine, cadaverine, spermidine, andspermine.

As used herein, “halogen” means Br, Cl, F and I.

In an embodiment of Formula A, R¹ and R² are independently selected fromH and (C₁-C₆)alkyl, wherein said alkyl is optionally substituted withone to three substituents selected from R′, or R¹ and R² can be takentogether with the nitrogen to which they are attached to form amonocyclic heterocycle with 4-7 members optionally containing, inaddition to the nitrogen, one or two additional heteroatoms selectedfrom N, O and S, said monocyclic heterocycle is optionally substitutedwith one to three substituents selected from R′.

In an embodiment of Formula A, R¹ and R² are independently selected fromH, methyl, ethyl and propyl, wherein said methyl, ethyl and propyl areoptionally substituted with one to three substituents selected from R′,or R¹ and R² can be taken together with the nitrogen to which they areattached to form a monocyclic heterocycle with 4-7 members optionallycontaining, in addition to the nitrogen, one or two additionalheteroatoms selected from N, O and S, said monocyclic heterocycle isoptionally substituted with one to three substituents selected from R′.

In an embodiment of Formula A, R¹ and R² are independently selected fromH, methyl, ethyl and propyl.

In an embodiment of Formula A, R¹ and R² are each methyl.

In an embodiment of Formula A, R′ is R″.

In an embodiment of Formula A, R″ is independently selected from H,methyl, ethyl and propyl, wherein said methyl, ethyl and propyl areoptionally substituted with one or more halogen and OH.

In an embodiment of Formula A, R″ is independently selected from H,methyl, ethyl and propyl.

In an embodiment of Formula A, L₁ is selected from C₄-C₂₂ alkyl andC₄-C₂₂ alkenyl, which are optionally substituted with halogen and OH.

In an embodiment of Formula A, L₁ is selected from C₄-C₂₂ alkyl andC₄-C₂₂ alkenyl.

In an embodiment of Formula A, L₁ is selected from C₄-C₂₂ alkenyl.

In an embodiment of Formula A, L₁ is selected from C₁₂-C₂₂ alkenyl.

In an embodiment of Formula A, L₁ is C₁₈ alkenyl.

In an embodiment of Formula A, L₁ is:

In an embodiment of Formula A, L₁ is:

In an embodiment of Formula A, L₂ is selected from C₃-C₁₃ alkyl andC₃-C₁₃ alkenyl, which are optionally substituted with halogen and OH.

In an embodiment of Formula A, L₂ is selected from C₃-C₉ alkyl and C₃-C₉alkenyl, which are optionally substituted with halogen and OH.

In an embodiment of Formula A, L₂ is selected from C₃-C₈ alkyl and C₃-C₈alkenyl, which are optionally substituted with halogen and OH.

In an embodiment of Formula A, L₂ is selected from C₃-C₁₃ alkyl andC₃-C₁₃ alkenyl.

In an embodiment of Formula A, L₂ is selected from C₃-C₉ alkyl and C₃-C₉alkenyl.

In an embodiment of Formula A, L₂ is selected from C₃-C₈ alkyl and C₃-C₈alkenyl.

In an embodiment of Formula A, L₂ is C₃-C₁₃ alkyl.

In an embodiment of Formula A, L₂ is C₃-C₉ alkyl.

In an embodiment of Formula A, L₂ is C₃-C₈ alkyl.

In an embodiment of Formula A, L₂ is C₈ alkyl.

In an embodiment of Formula A, “heterocyclyl” is pyrolidine, piperidine,morpholine, imidazole or piperazine.

In an embodiment of Formula A, “monocyclic heterocyclyl” is pyrolidine,piperidine, morpholine, imidazole or piperazine.

In an embodiment of Formula A, “polyamine” is putrescine, cadaverine,spermidine or spermine.

In an embodiment, “alkyl” is a straight chain saturated aliphatichydrocarbon having the specified number of carbon atoms.

In an embodiment, “alkenyl” is a straight chain unsaturated aliphatichydrocarbon having the specified number of carbon atoms.

Included in the instant invention is the free form of cationic lipids ofFormula A, as well as the pharmaceutically acceptable salts andstereoisomers thereof. Some of the isolated specific cationic lipidsexemplified herein are the protonated salts of amine cationic lipids.The term “free form” refers to the amine cationic lipids in non-saltform. The encompassed pharmaceutically acceptable salts not only includethe isolated salts exemplified for the specific cationic lipidsdescribed herein, but also all the typical pharmaceutically acceptablesalts of the free form of cationic lipids of Formula A. The free form ofthe specific salt cationic lipids described may be isolated usingtechniques known in the art. For example, the free form may beregenerated by treating the salt with a suitable dilute aqueous basesolution such as dilute aqueous NaOH, potassium carbonate, ammonia andsodium bicarbonate. The free forms may differ from their respective saltforms somewhat in certain physical properties, such as solubility inpolar solvents, but the acid and base salts are otherwisepharmaceutically equivalent to their respective free forms for purposesof the invention.

The pharmaceutically acceptable salts of the instant cationic lipids canbe synthesized from the cationic lipids of this invention which containa basic or acidic moiety by conventional chemical methods. Generally,the salts of the basic cationic lipids are prepared either by ionexchange chromatography or by reacting the free base with stoichiometricamounts or with an excess of the desired salt-forming inorganic ororganic acid in a suitable solvent or various combinations of solvents.Similarly, the salts of the acidic compounds are formed by reactionswith the appropriate inorganic or organic base.

Thus, pharmaceutically acceptable salts of the cationic lipids of thisinvention include the conventional non-toxic salts of the cationiclipids of this invention as formed by reacting a basic instant cationiclipids with an inorganic or organic acid. For example, conventionalnon-toxic salts include those derived from inorganic acids such ashydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric andthe like, as well as salts prepared from organic acids such as acetic,propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,trifluoroacetic (TFA) and the like.

When the cationic lipids of the present invention are acidic, suitable“pharmaceutically acceptable salts” refers to salts prepared formpharmaceutically acceptable non-toxic bases including inorganic basesand organic bases. Salts derived from inorganic bases include aluminum,ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganicsalts, manganous, potassium, sodium, zinc and the like. Particularlypreferred are the ammonium, calcium, magnesium, potassium and sodiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as arginine, betainecaffeine, choline, N,N¹-dibenzylethylenediamine, diethylamin,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylaminetripropylamine, tromethamine and the like.

The preparation of the pharmaceutically acceptable salts described aboveand other typical pharmaceutically acceptable salts is more fullydescribed by Berg et al., “Pharmaceutical Salts,” J Pharm. Set.,1977:66:1-19.

It will also be noted that the cationic lipids of the present inventionare potentially internal salts or zwitterions, since under physiologicalconditions a deprotonated acidic moiety in the compound, such as acarboxyl group, may be anionic, and this electronic charge might then bebalanced off internally against the cationic charge of a protonated oralkylated basic moiety, such as a quaternary nitrogen atom.

EXAMPLES

Examples provided are intended to assist in a further understanding ofthe invention. Particular materials employed, species and conditions areintended to be further illustrative of the invention and not limitativeof the reasonable scope thereof. The reagents utilized in synthesizingthe cationic lipids are either commercially available or are readilyprepared by one of ordinary skill in the art.

Synthesis of the novel cationic lipids is a linear process startingepichlorohydrin (1). Reaction of epichlorohydrin with lipid alcoholgives intermediate epoxide ii. Lewis acid epoxide opening with thesecond lipid alcohol affords secondary alcohol iii. Triflate formationand amine substitution gives final products of type iv.

Synthesis of intermediate cyclopropyl analogs (vi) was conducted bySimmons-Smith cyclopropanation of olefins of type v.

A solution of octanol (1.5 mol, 195 g), tetrabutyl ammonium bromide (75mmol, 24.1 g) and sodium hydroxide (2.4 mol, 96 g) was cooled to 3 C andR-epichlorohydrin was added via dropping funnel. The mixture was stirredat 3 C for 6 hours and then allowed to warm to ambient temperature for16 hours. The reaction was partitioned between hexanes and aqueoussaturated sodium bicarbonate. The organics were washed with water (2×625mL) and brine (625 mL) and evaporated in vacuo. The crude oil waspurified by flash chromatography (silica, 0-20% ethyl acetate/heptane)to give 236 g (85%) of one of the enantiomers of2-[(octyloxy)methyl]oxirane as a clear oil. ¹H NMR (400 MHz, CDCl3): δ3.68 (m, 1H), 3.45 (m, 2H), 3.38 (m, 1H), 3.16 (m, 1H), 2.78 (m, 1H),2.59 (m, 1H), 1.58 (m, 2H), 1.30 (m, 10H), 0.83 (m, 3H).

The product of this reaction was first assigned the 2R stereochemistry,which would be obtained via a SN2 mechanism at the carbon bearing thehalide atom with no change at the asymmetric carbon centre.

However, subsequent studies (experiments on the reaction of lineolylalcohol with S-epichlorohydrin), under conditions described, Lewis acidconditions and in conjunction with vibrational circular dichroism (VCD),a valid spectroscopic method for determining absolute configuration ofchiral molecules (R. K. Dukor and L. A. Nafie, in Encyclopedia ofAnalytical Chemistry: Instrumentation and Applications, Ed. R. A. Meyers(Wiley, Chichester, 2000) 662-676) have revealed the reaction occurredvia a SN2′ mechanism ie reaction at the terminal carbon of the epoxideleading to subsequent ring opening, followed by an in situ ring closingstep that leads to inversion of stereochemistry at the asymmetriccarbon.

As a result of these studies, the product from this reaction wasreassigned the 2S stereochemistry: (2S)-[(octyloxy)methyl]oxirane.

A solution of epoxide (3.86 g, 20.7 mmol) and linoleyl alcohol (6.6 g,24.8 mmol) in dichloromethane (100 mL) was treated with tintetrachloride (4.44 g, 2.0 mmol) at ambient temperature. After stirringfor 3 hours, the reaction was quenched with brine and partitionedbetween brine and dichloromethane. The organics were dried over sodiumsulfate, filtered and evaporated in vacuo. The crude oil was purified byflash chromatography (silica, 2-5% acetone/hexanes) to give 5.85 g (62%)of R-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-ol asa clear oil. ¹H NMR (400 MHz, CDCl3): δ 5.38 (m, 4H), 3.93 (m, 1H), 3.49(m, 8H), 2.79 (m, 2H), 2.45 (m, 1H), 2.06 (m, 4H), 1.58 (m, 6H), 1.30(m, 26H), 0.89 (m, 6H).

Compound iii (5.08 g, 11.2 mmol) was dissolved into dichloromethane (100ml) and treated with 2,6-lutidine (1.44 g, 13.5 mmol). Triflic anhydride(3.8 g, 13.5 mmol) was added dropwise into the reaction system at 0° C.After 2.5 hours, the reaction was cannulated into a cold solution ofdimethylamine (39.3 mmol) in dichloromethane (85 mL). After 2 hours at 0C, the reaction was quenched with saturated sodium bicarbonate solutionand partitioned between water/dichloromethane. The organics were driedover magnesium sulfate, filtered and evaporated in vacuo. The crude oilwas purified by flash chromatography (silica, 0-30% ethylacetate/hexanes) to give 3.66 g (65%) ofS—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amineas an amber oil. ¹H NMR (400 MHz, CDCl3): δ 5.38 (m, 4H), 3.52 (m, 4H),3.40 (m, 4H), 2.79 (m, 3H), 2.40 (s, 6H), 2.05 (m, 4H), 1.58 (m, 4H),1.30 (m, 26H), 0.89 (m, 6H). HRMS: cal'd-480.4775. found 480.4769.

Compound 2 is a novel cationic lipid and was prepared according to theGeneral Scheme 1 and Scheme 1 above, starting with S-epichlorohydrin.

R—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine(Compound 2)

¹H NMR (400 MHz, CDCl3): δ 5.38 (m, 4H), 3.52 (m, 4H), 3.40 (m, 4H),2.79 (m, 311), 2.40 (s, 6H), 2.05 (m, 4H), 1.58 (m, 4H), 1.30 (m, 2611),0.89 (m, 6H).

Compounds 3-20 were prepared in a manner analogous to that described forCompound 1 according to General Scheme 1.

HRMS/LCM Compound Name Structure S (M + H) 3 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1- yloxy]-1- ((octyloxy)methyl] ethyl}pyrrolidine

C33H63NO2 [M + H] calc 506.5 obs 506.7 4 (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadeca- 9,12-dien-1-yloxy]-3- [(5Z)-oct-5-en-1-yloxy]propan-2-amine

C31H59NO2 [M + H] calc 478.5 obs 478.5 5 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1- yloxy]-1- [(octyloxy)methyl] ethyl}azetidine

C32H61NO2 [M + H] calc 492.5 obs 492.7 6 (2S)-1-(hexyloxy)-N,N-dimethyl-3- [(9Z,12Z)-octadeca- 9,12-dien-1- yloxylpropan-2-amine

C29H57NO2 [M + H] calc 452.4 obs 452.4 7 (2S)-1-(heptyloxy)-N,N-dimethyl-3- [(9Z,12Z)-octadeca- 9,12-dien-1- yloxy]propan-2-amine

C30H59NO2 [M + H] calc 466.5 obs 466.5 8 N,N-dimethyl-1- (nonyloxy)-3-[(9Z,12Z)-octadeca- 9,12-dien-1- yloxy]propan-2-amine

C32H63NO2 [M + H] calc 494.5 obs 494.5 9 N,N-dimethyl-1-[(9Z)-octadec-9-en-1- yloxy]-3- (octyloxy)propan-2- amine

C31H63NO2 [M + H] calc 482.5 obs 482.5 10 (2S)-N,N-dimethyl-1-[(6Z,9Z,12Z)- octadeca-6,9,12-trien- 1-yloxy]-3- (octyloxy)propan-2-amine

C31H59NO2 [M + H] calc 478.5 obs 478.5 11 (2S)-1-[(11Z,14Z)-icosa-11,14-dien-1- yloxy]-N,N-dimethyl- 3-(pentyloxy)propan- 2-amine

C30H59NO2 [M + H] calc 466.5 obs 466.5 12 (2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa- 11,14-dien-1-yloxy]- N,N-dimethylpropan- 2-amine

C31H61NO2 [M + H] calc 480.5 obs 480.5 13 1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]- N,N-dimethyl-3- (octyloxy)propan-2- amine

C33H65NO2 [M + H] calc 508.5 obs 508.5 14 1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]- N,N-dimethyl-3- (octyloxy)propan-2- amine

C35H69NO2 [M + H] calc 536.5 obs 536.5 15 (2S)-1-[(13Z,16Z)-docosa-13,16-dien-1- yloxy]-3-(hexyloxy)- N,N-dimethylpropan- 2-amine

C33H65NO2 [M + H] calc 508.5 obs 508.5 16 (2S)-1-[(13Z)-docos-13-en-1-yloxy]-3- (hexyloxy)-N,N- dimethylpropan-2- amine

C33H67NO2 [M + H] calc 510.5 obs 510.5 17 1-[(13Z)-docos-13-en-1-yloxy]-N,N- dimethyl-3- (octyloxy)propan-2- amine

C35H71NO2 [M + H] calc 538.6 obs 538.6 18 1-((9Z)-hexadec-9-en-1-yloxy]-N,N- dimethyl-3- (octyloxy)propan-2- amine

C29H59NO2 [M + H] calc 454.5 obs 454.5 19 (2R)-N,N-dimethyl-1-[(1-methyloctyl)oxy]- 3-[(9Z,12Z)-octadeca- 9,12-dien-1-yloxy]propan-2-amine

C32H63NO2 [M + H] calc 494.5 obs 494.7 20 (2R)-1-(3,7-dimethyloctyl)oxy]- N,N-dimethyl-3- [(9Z,12Z)-octadeca- 9,12-dien-1-yloxy]propan-2-amine

C33H65NO2 [M + H] calc 508.5 obs 508.5

A solution of1-[(9Z,12Z)-oetadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-ol in DCM(166 ml) in a 1 L flask under nitrogen was cooled in an ice/IPA bath(−15° C.) and then 1 M diethylzine in hexane (199 ml, 199 mmol) added,followed by diiodomethane (24.05 ml, 298 mmol). The mixture was allowedto stir in the cooling bath overnight to gradually come up to roomtemperature. Reaction was quenched by addition of excess saturatedammonium chloride. Washed reaction into 2 L separatory funnel withhexane and water and added excess hexane, shook vigorously. Discardedlower aqueous layer, dried organic layer with sodium sulfate andevaporated to1-(octyloxy)-3-[(8-{2-[(2-pentylcyclopropyl)methyl]cyclopropyl}octyl)oxy]propan-2-ol(15.5 g, 32.2 mmol, 97% yield) as a yellow oil. Material was used withno further purification. MS (ESI): 481 [M+H]+.

Conversion of alcohol vi to final compound 21 was accomplished asdescribed for Compound 1. C33H65NO2 [M+H], calc 508.5, obs 508.5.

N,N-dimethyl-1-{[8-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine(Compound 22) was prepared in a manner analogous to that described forcompound 21. C32H65NO2 [M+H] calc 496.5, obs 496.5.

Compound 23 is DLinDMA as described in J. Controlled Release, 2005, 107,276-287, US 2006/0083780 A1, and US 2006/0008910 A1.

Compound 24 is DLinKC2DMA as described in Nature Biotechnology, 2010,28, 172-176, WO 2010/042877 A1, WO 2010/048536 A2, WO 2010/088537 A2,and WO 2009/127060 A1.

LNP COMPOSITIONS

The following lipid nanoparticle compositions (LNPs) of the instantinvention are useful for the delivery of oligonucleotides, specificallysiRNA and miRNA:

-   Cationic Lipid/Cholesterol/PEG-DMG 56.6/38/5.4;-   Cationic Lipid/Cholesterol/PEG-DMG 60/38/2;-   Cationic Lipid/Cholesterol/PEG-DMG 67.3/29/3.7;-   Cationic Lipid/Cholesterol/PEG-DMG 493/47/3.7;-   Cationic Lipid/Cholesterol/PEG-DMG 503/44.3/5.4;-   Cationic Lipid/Cholesterol PEG-C-DMA/DSPC 40/48/2/10;-   Cationic Lipid/Cholesterol/PEG-DMG/DSPC 40/48/2/10; and-   Cationic Lipid/Cholesterol/PEG-DMG DSPC 58/30/2/10.

The synthesis and use of LNPs are known. (See US patent applications: US2006/0083780, US 2006/0240554, US 2008/0020058, US 2009/0263407 and US2009/0285881 and PCT patent applications: WO 2009/086558, WO2009/127060,WO2009/132131, WO2010/042877, WO2010/054384, WO2010/054401,WO2010/054405 and WO2010/054406). See also Semple S. C. et al., Rationaldesign of cationic lipids for siRNA delivery, Nature Biotechnology,published online 17 Jan. 2010;

doi:10.1038/nbt.1602.

LNP Process Description:

The Lipid Nano-Particles (LNP) are prepared by an impinging jet process.The particles are formed by mixing lipids dissolved in alcohol withsiRNA dissolved in a citrate buffer. The mixing ratio of lipids to siRNAare targeted at 45-55% lipid and 65-45% siRNA. The lipid solutioncontains a novel cationic lipid of the instant invention, a helper lipid(cholesterol), PEG (e.g. PEG-C-DMA, PEG-DMG) lipid, and DSPC at aconcentration of 5-15 mg/mL with a target of 9-12 mg/mL in an alcohol(for example ethanol). The ratio of the lipids has a mole percent rangeof 25-98 for the cationic lipid with a target of 35-65, the helper lipidhas a mole percent range from 0-75 with a target of 30-50, the PEG lipidhas a mole percent range from 1-15 with a target of 1-6, and the DSPChas a mole precent range of 0-15 with a target of 0-12. The siRNAsolution contains one or more siRNA sequences at a concentration rangefrom 0.3 to 1.0 mg/mL with a target of 0.3-0.9 mg/mL in a sodium citratebuffered salt solution with pH in the range of 3.5-5. The two liquidsare heated to a temperature in the range of 15-40° C., targeting 30-40°C., and then mixed in an impinging jet mixer instantly forming the LNP.The teeID has a range from 0.25 to 1.0 mm and a total flow rate from10-600 mL/min. The combination of flow rate and tubing ID has effect ofcontrolling the particle size of the LNPs between 30 and 200 nm. Thesolution is then mixed with a buffered solution at a higher pH with amixing ratio in the range of 1:1 to 1:3 vol:vol but targeting 1:2vol:vol. This buffered solution is at a temperature in the range of15-40° C., targeting 30-40° C. The mixed LNPs are held from 30 minutesto 2 hrs prior to an anion exchange filtration step. The temperatureduring incubating is in the range of 15-40° C., targeting 30-40° C.After incubating the solution is filtered through a 0.8 urn filtercontaining an anion exchange separation step. This process uses tubingIDs ranging from 1 mm ID to 5 mm ID and a flow rate from 10 to 2000mL/min. The LNPs are concentrated and diafiltered via an ultrafiltrationprocess where the alcohol is removed and the citrate buffer is exchangedfor the final buffer solution such as phosphate buffered saline. Theultrafiltration process uses a tangential flow filtration format (TFF).This process uses a membrane nominal molecular weight cutoff range from30-500 KD. The membrane format can be hollow fiber or flat sheetcassette. The TFF processes with the proper molecular weight cutoffretains the LNP in the retentate and the filtrate or permeate containsthe alcohol; citrate buffer; final buffer wastes. The TFF process is amultiple step process with an initial concentration to a siRNAconcentration of 1-3 mg/mL. Following concentration, the LNPs solutionis diafiltered against the final buffer for 10-20 volumes to remove thealcohol and perform buffer exchange. The material is then concentratedan additional 1-3 fold. The final steps of the LNP process are tosterile filter the concentrated LNP solution and vial the product.

Analytical Procedure:

1) siRNA Concentration

The siRNA duplex concentrations are determined by Strong Anion-ExchangeHigh-Performance Liquid Chromatography (SAX-HPLC) using Waters 2695Alliance system (Water Corporation, Milford Mass.) with a 2996 PDAdetector. The LNPs, otherwise referred to as RNAi Delivery Vehicles(RDVs), are treated with 0.5% Triton X-100 to free total siRNA andanalyzed by SAX separation using a Dionex BioLC DNAPac PA 200 (4×250 mm)column with UV detection at 254 nm. Mobile phase is composed of A: 25 mMNaClO₄, 10 mM Tris, 20% EtOH, pH 7.0 and B: 250 mM NaClO₄, 10 mM Tris,20% EtOH, pH 7.0 with liner gradient from 0-15 min and flow rate of 1ml/min. The siRNA amount is determined by comparing to the siRNAstandard curve.

2) Encapsulation Rate

Fluorescence reagent SYBR Gold is employed for RNA quantitation tomonitor the encapsulation rate of RDVs. RDVs with or without TritonX-100 are used to determine the free siRNA and total siRNA amount. Theassay is performed using a SpectraMax M5e microplate spectrophotometerfrom Molecular Devices (Sunnyvale, Calif.). Samples are excited at 485nm and fluorescence emission was measured at 530 nm. The siRNA amount isdetermined by comparing to the siRNA standard curve.

Encapsulation rate=(1−free siRNA/total siRNA)×100%

3) Particle Size and Polydispersity

RDVs containing 1 μg siRNA are diluted to a final volume of 3 ml with1×PBS. The particle size and polydispersity of the samples is measuredby a dynamic light scattering method using ZetaPALS instrument(Brookhaven Instruments Corporation, Holtsville, N.Y.). The scatteredintensity is measured with HeNe laser at 25° C. with a scattering angleof 90°.

4) Zeta Potential Analysis

RDVs containing 1 μg siRNA are diluted to a final volume of 2 ml with 1mM Tris buffer (pH 7.4). Electrophoretic mobility of samples isdetermined using ZetaPALS instrument (Brookhaven InstrumentsCorporation, Holtsville, N.Y.) with electrode and He—Ne laser as a lightsource. The Smoluchowski limit is assumed in the calculation of zetapotentials.

5) Lipid Analysis

Individual lipid concentrations are determined by Reverse PhaseHigh-Performance Liquid Chromatography (RP-HPLC) using Waters 2695Alliance system (Water Corporation, Milford Mass.) with a Corona chargedaerosol detector (CAD) (ESA Biosciences, Inc, Chelmsford, Mass.).Individual lipids in RDVs are analyzed using an Agilent Zorbax SB-C18(50×4.6 mm, 1.8 μm particle size) column with CAD at 60° C. The mobilephase is composed of A: 0.1% TFA in H₂O and B: 0.1% TFA in IPA. Thegradient changes from 60% mobile phase A and 40% mobile phase B fromtime 0 to 40% mobile phase A and 60% mobile phase B at 1.00 min; 40%mobile phase A and 60% mobile phase B from 1.00 to 5.00 min; 40% mobilephase A and 60% mobile phase B from 5.00 min to 25% mobile phase A and75% mobile phase B at 10.00 min; 25% mobile phase A and 75% mobile phaseB from 10.00 min to 5% mobile phase A and 95% mobile phase B at 15.00min; and 5% mobile phase A and 95% mobile phase B from 15.00 to 60%mobile phase A and 40% mobile phase B at 20.00 min with flow rate of 1nil/min. The individual lipid concentration is determined by comparingto the standard curve with all the lipid components in the RDVs with aquadratic curve fit. The molar percentage of each lipid is calculatedbased on its molecular weight.

Utilizing the above described LNP process, specific LNPs with thefollowing ratios were identified:

Nominal Composition:

-   Cationic Lipid/Cholesterol/PEG-DMG 60/38/2-   Cationic Lipid/Cholesterol/PEG-DMG 67.3/29/3.7    Luc siRNA

(SEQ.ID.NO.: 1) 5′-iB-A U AAGG CU A U GAAGAGA U ATT-iB 3′(SEQ.ID.NO.: 2) 3′-UUUAUUCCGAUACUUCUC UAU-5′ AUGC-RiboseiB-Inverted deoxy abasic UC-2′ Fluoro AGT-2′ Deoxy AGU-2′ OCH₃

Nominal Composition

-   Cationic Lipid/Cholesterol/PEG-DMG 60/38/2-   Cationic Lipid/Cholesterol/PEG-DMG/DSPC 40/48/2/10-   Cationic Lipid/Cholesterol/PEG-DMG/DSPC 58/30/2/10    ApoB siRNA

(SEQ ID NO.: 3) 5′-iB-CUUU AA C AA UUCCU GAAA U TT-iB  (SEQ ID NO.: 4)3′-UUGAAAUUGUUAAGGACU UUA-5′ AUGC-Ribose iB-Inverted deoxy abasic UC-2′Fluoro AGT-2′ Deoxy AGU-2′ OCH₃

Nominal Composition

-   Cationic Lipid/Cholesterol/PEG-DMG 60/38/2-   Cationic Lipid/Cholesterol/PEG-DMG/DSPC 40/48/2/10-   Cationic Lipid/Cholesterol/PEG-DMG DSPC 58/30/2/10    ApoB siRNA

(SEQ ID NO.: 5) 5′-iB-CUUUAACAAUUCCUGAAAUTsT-iB-3′ (SEQ ID NO.: 6)3′-UsUGAAAUUGUUAAGGACUsUsUsA-5′ AUGC-Ribose iB-Inverted deoxy abasicUC-2′ Fluoro AGT-2′ Deoxy AGU-2′ OCH₃ UsA-phophorothioate linkage

The synthesis and use of oligonucleotides, in particular siRNA andmiRNA, are known. (See US patent applications: US 2006/0083780, US2006/0240554, US 2008/0020058, US 2009/0263407 and US 2009/0285881 andPCT patent applications: WO 2009/086558, WO2009/127060, WO2009/132131,WO2010/042877, WO2010/054384, WO2010/054401, WO2010/054405 andWO2010/054406). See also Semple S. C. et al., Rational design ofcationic lipids for siRNA delivery, Nature Biotechnology, publishedonline 17 Jan. 2010; doi:10.1038/nbt.1602.

Example 1 Mouse In Vivo Evaluation of Efficacy

LNPs utilizing Compounds 1-2, in the nominal compositions describedimmediately above, were evaluated for in vivo efficacy and induction ofinflammatory cytokines in a luciferase mouse model. The siRNA targetsthe mRNA transcript for the firefly (Photinus pyralis) luciferase gene(Accession # M15077). The primary sequence and chemical modificationpattern of the luciferase siRNA is displayed above. The in vivoluciferase model employs a transgenic mouse in which the fireflyluciferase coding sequence is present in all cells.ROSA26-LoxP-Stop-LoxP-Luc (LSL-Luc) transgenic mice licensed from theDana Farber Cancer Institute are induced to express the Luciferase geneby first removing the LSL sequence with a recombinant Ad-Cre virus(Vector Biolabs). Due to the organo-tropic nature of the virus,expression is limited to the liver when delivered via tail veininjection. Luciferase expression levels in liver are quantitated bymeasuring light output, using an IVIS imager (Xenogen) followingadministration of the luciferin substrate (Caliper Life Sciences).Pre-dose luminescence levels are measured prior to administration of theRDVs. Luciferin in PBS (15 mg/mL) is intraperitoneally (IP) injected ina volume of 150 μL. After a four minute incubation period mice areanesthetized with isoflurane and placed in the IVIS imager. The RDVs(containing siRNA) in PBS vehicle were tail vein injected n a volume of0.2 mL. Final dose levels ranged from 0.3 to 3 mg/kg siRNA. PBS vehiclealone was dosed as a control. Three hours post dose, mice were bledretro-orbitally to obtain plasma for cytokine analysis. Mice were imaged48 hours post dose using the method described above. Changes inluciferin light output directly correlate with luciferase mRNA levelsand represent an indirect measure of luciferase siRNA activity. In vivoefficacy results are expressed as % inhibition of luminescence relativeto pre-dose luminescence levels. Plasma cytokine levels were determinedusing the SearchLight multiplexed cytokine chemoluminescent array(Pierce/Thermo). Systemic administration of the luciferase siRNA RDVsdecreased luciferase expression in a dose dependant manner. Greaterefficacy was observed in mice dosed with Compound 1 containing RDVs thanwith the RDV containing the octyl-CLinDMA (OCD) cationic lipid (FIG. 1).OCD is known and described in WO2010/021865.

Rat In Vivo Evaluation of Efficacy and Toxicity

LNPs utilizing Compound 1 in the nominal compositions described above,were evaluated for in vivo efficacy and increases in alanine aminotransferase and aspartate amino transferase in Sprague-Dawley(Crl:CD(SD) female rats (Charles River Labs). The siRNA targets the mRNAtranscript for the ApoB gene (Accession # NM 019287). The primarysequence and chemical modification pattern of the ApoB siRNA isdisplayed above. The RDVs (containing siRNA) in PBS vehicle were tailvein injected in a volume of 1 to 1.5 mL. Infusion rate is approximately3 ml/min. Five rats were used in each dosing group. After LNPadministration, rats are placed in cages with normal diet and waterpresent. Six hours post dose, food is removed from the cages. Animalnecropsy is performed 24 hours after LNP dosing. Rats are anesthetizedunder isoflurane for 5 minutes, then maintained under anesthesia byplacing them in nose cones continuing the delivery of isoflurane untilex-sanguination is completed. Blood is collected from the vena cavausing a 23 guage butterfly venipuncture set and aliquoted to serumseparator vacutainers for serum chemistry analysis. Punches of theexcised caudate liver lobe are taken and placed in RNALater (Ambion) formRNA analysis. Preserved liver tissue was homogenized and total RNAisolated using a Qiagen bead mill and the Qiagen miRNA-Easy RNAisolation kit following the manufacturer's instructions. Liver ApoB mRNAlevels were determined by quantitative RT-PCR, Message was amplifiedfrom purified RNA utilizing a rat ApoB commercial probe set (AppliedBiosystems Cat # RN01499054_ml). The PCR reaction was performed on anABI 7500 instrument with a 96-well Fast Block. The ApoB mRNA level isnormalized to the housekeeping PPIB (NM 011149) mRNA. PPIB mRNA levelswere determined by RT-PCR using a commercial probe set (AppliedBiosytems Cat. No. Mm00478295_ml). Results are expressed as a ratio ofApoB mRNA/PPIB mRNA. All mRNA data is expressed relative to the PBScontrol dose. Serum ALT and AST analysis were performed on the SiemensAdvia 1800 Clinical Chemistry Analyzer utilizing the Siemens alanineaminotransferase (Cat#03039631) and aspartate aminotransferase(Cat#03039631) reagents. Greater efficacy was observed in rats dosedwith Compound 1 containing RDV than with the RDV containing theoctyl-CLinDMA cationic lipid (FIG. 2). Additionally, lower elevations inLFTs (ALT/AST) were observed in rats dosed with Compound 1 containingRDV than with the RDV containing the octyl-CLinDMA cationic lipid (FIG.3).

Determination of Cationic Lipid Levels in Rat Liver

Liver tissue was weighed into 20-ml vials and homogenized in 9 v/w ofwater using a GenoGrinder 2000 (OPS Diagnostics, 1600 strokes/min, 5min). A 50 μL aliquot of each tissue homogenate was mixed with 300 μL ofextraction/protein precipitating solvent (50/50 acetonitrile/methanolcontaining 500 nM internal standard) and the plate was centrifuged tosediment precipitated protein. A volume of 2004 of each supernatant wasthen transferred to separate wells of a 96-well plate and 10 μl sampleswere directly analyzed by LC/MS-MS.

Standards were prepared by spiking known amounts of a methanol stocksolution of Compound 1 or OCD into untreated rat liver homogenate (9 volwater/weight liver). Aliquots (50 μL) each standard/liver homogenate wasmixed with 300 μL of extraction/protein precipitating solvent (50/50acetonitrile/methanol containing 500 nM internal standard) and the platewas centrifuged to sediment precipitated protein. A volume of 200 pa, ofeach supernatant was transferred to separate wells of a 96-well plateand 10 μl of each standard was directly analyzed by LC/MS-MS.

Absolute quantification versus standards prepared and extracted from ratliver homogenate was performed using an Aria LX-2 HPLC system (ThermoScientific) coupled to an API 4000 triple quadrupole mass spectrometer(Applied Biosystems). For each run, a total of 10 mL sample was injectedonto a BDS Hypersil C8 HPLC column (Thermo, 50×2 mm, 3 μm) at ambienttemperature (FIG. 4).

Mobile Phase A: 95% H2O/5% methanol/10 mM ammonium formate/0.1% formicacid Mobile Phase B: 40% methanol/60% n-propanol/10 mM ammoniumformate/0.1% formic acid The flow rate was 0.5 mL/min and gradientelution profile was as follows: hold at 80% A for 0.25 min, linear rampto 100% B over 1.6 min, hold at 100% B for 2.5 min, then return and holdat 80% A for 1.75 min. Total run time was 5.8 min. API 4000 sourceparameters were CAD: 4, CUR: 15, OSI: 65, GS2: 35, IS: 4000, TEM: 550,CXP: 15, DP: 60, EP: 10.

What is claimed is:
 1. A cationic lipid of Formula A:

wherein: R¹ and R² are independently selected from H, (C₁-C₆)alkyl,heterocyclyl, and polyamine, wherein said alkyl, heterocyclyl andpolyamine are optionally substituted with one to three substituentsselected from R′, or R¹ and R² can be taken together with the nitrogento which they are attached to form a monocyclic heterocycle with 4-7members optionally containing, in addition to the nitrogen, one or twoadditional heteroatoms selected from N, O and S, said monocyclicheterocycle is optionally substituted with one to three substituentsselected from R¹; R′ is independently selected from halogen, R″, OR″,SR″, CN, CO₂R″ or CON(R″)₂; R″ is independently selected from H and(C₁-C₆)alkyl, wherein said alkyl is optionally substituted with halogenand OH; L₁ is selected from C₄-C₂₂ alkyl and C₄-C₂₂ alkenyl, said alkyland alkenyl are optionally substituted with one or more substituentsselected from R′; and L₂ is selected from C₃-C₁₃ alkyl and C₃-C₁₃alkenyl, said alkyl and alkenyl are optionally substituted with one ormore substituents selected from R′; or any pharmaceutically acceptablesalt or stereoisomer thereof.
 2. A cationic lipid of Formula A accordingto claim 1, wherein: R¹ and R² are each methyl; L₁ is selected fromC₄-C₂₂ alkyl and C₄-C₂₂ alkenyl; and L₂ is selected from C₃-C₁₃ alkyland C₃-C₁₃ alkenyl; or any pharmaceutically acceptable salt orstereoisomer thereof.
 3. A cationic lipid which is selected from:R—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine(Compound 2);S—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine(Compound 1);1-{2-[(9412Z)-octadeca-9,12-dien-1-yloxy]-1-[(actyloxy)methyl]ethyl}pyrrolidine(Compound 3);(2S)—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine(Compound 4);1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine(Compound 5);(2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine(Compound 6);(2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine(Compound 7);N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine(Compound 8);N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-amine(Compound 9); (2 S)—N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine(Compound 10);(2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine(Compound 11); (2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine(Compound 12);1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine(Compound 13);1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine(Compound 14); (2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine(Compound 15);(2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine(Compound 16);1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine(Compound 17);1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine(Compound 18);(2R)—N,N-dimethyl-1-[(1-methyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine(Compound 19);(2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine(Compound 20);N,N-dimethyl-1-(octyloxy)-3-((8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyloxy)propan-2-amine(Compound 21); andN,N-dimethyl-1-{[8-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine(Compound 22); or any pharmaceutically acceptable salt or stereoisomerthereof.
 4. The use of a cationic lipid according to claim 1 for thepreparation of lipid nanoparticles.
 5. The use of a cationic lipidaccording to claim 1 as a component in a lipid nanoparticle for thedelivery of oligonucleotides.
 6. The use according to claim 5 whereinthe oligonucleotides are siRNA or miRNA.
 7. The use according to claim 5wherein the oligonucleotides are siRNA.